Thiolation can be used to introduces the possibility of specific coupling to a sulfydryl-containing ligand through a disulfide bridge. The terminal thiol group allows attachment of an oligo to various ligands, such as biotin, alkaline phosphatase or fluorescent dyes, via maleimide, iodide, bromide, or sulphonyl derivatives. Additionally, the terminal thiol group allows attachment of an oligo to solid surfaces via an iodoacetamide or maleimide linkage. Thiol modification can be placed at either the 5' or 3' end of the oligonucleotide.
Thiol-Modifier C3 S-S
The thiol group is protected by a disulphate bond following synthesis of the oligo and must be deprotected by a reducing agent before use. The reducing agent should be completely removed immediately before the oligo is used in a coupling reaction. DTT is most commonly employed as a reducing agent.
Thiol modifier C3 S-S (5')
Protocol for thiol modifier deprotection
- Cleave the disulfide linkage at 37°C in DTT (100 mM, pH 8.3) for approximately 30 min.
- Desalt the oligo on a column.
- Thiol-modified oligos that have been deprotected should be kept either under an inert atmosphere or in a solution containing DTT (10 mM) to avoid oxidative disulfide formation.
- The DTT should be removed completely by ethyl acetate extraction or by desalting before using the oligo.
Biolegio uses 3'-Thiol-Modifier C3 S-S CPG for 3'-labeling, and Thiol-Modifier C6 for 5'- labeling
Thiol Modifier C6 (5')